Developmental evidence helps resolve the evolutionary origins of anther appendages in Globba (Zingiberaceae) By: Limin Cao, Mark F. Newman, Bruce K. Kirchoff, and Louis P. Ronse de Craene This is a pre-copyedited, author-produced version of an article accepted for publication in Botanical Journal of the Linnean Society following peer review. The version of record, Limin Cao, Mark F Newman, Bruce K Kirchoff, Louis P Ronse de Craene; Developmental evidence helps resolve the evolutionary origins of anther appendages in Globba (Zingiberaceae), Botanical Journal of the Linnean Society, Volume 189, Issue 1, 1 January 2019, Pages 63–82. is available online at: https://doi.org/10.1093/botlinnean/boy071 ***© 2018 The Linnean Society of London. Reprinted with permission. No further reproduction is authorized without written permission from Oxford University Press. This version of the document is not the version of record. Figures and/or pictures may be missing from this format of the document. *** Abstract: Globba is one of the largest genera in the primarily tropical Zingiberaceae. The number of anther appendages is highly diagnostic and has been used along with molecular characters to define subgenera and sections. Four main types of anther morphology are recognized: without appendages and with two, four and six appendages. The six-appendaged anthers are reported here for the first time. Appendages arise from two dorsal ledges that flank the broad connective. Development of two-appendaged and four-appendaged species differs from inception. Previous suggestions that either the proximal or distal appendages of four-appendaged anthers have been lost in two-appendaged species are thus not supported. Early development of six-appendaged anthers is similar to that of four-appendaged species, but two additional, small appendages develop on the ledges between the first-formed appendages. This yields three appendages on each side (six overall). The four appendages of G. geoffrayi differ from all other species in having distal appendages that are much smaller and develop later than the proximal appendages. Development thus suggests that the state in G. geoffrayi evolved from a two-appendaged ancestor. Incorporating this information into a phylogenetic character plot of the number of appendages shows that the possession of two appendages is the most likely plesiomorphic state of the genus, although support for this hypothesis is weak. Our study clarifies the origin and complexity in the development of anther appendages in Globba and highlights their significance in infrageneric relationships in Globba. Two appendages have probably likely arisen at the base of Globba, linked with the presence of a prominent ledge, with variable extensions and reductions of the number of appendages in the various subgenera and sections. Keywords: connective | floral development | Globbeae | phylogeny Article: INTRODUCTION Globba L. is one of the largest genera in the primarily tropical Zingiberaceae and comprises c. 100 species distributed throughout tropical (and parts of subtropical) South-East Asia (Leong- Škorničková & Newman, 2015). The distribution of the genus ranges from India to southern China, south and east to the Philippines and New Guinea (Schumann, 1904). Globba [including Mantisia (Sims) K.J. Williams], Gagnepainia K.Schum. (three species) and Hemiorchis Kurz (three species) comprise Globbeae, one of the four traditionally recognized tribes of Zingiberaceae (e.g. Schumann, 1904; Larsen et al., 1998; Williams, Kress & Manos, 2004). Kress, Prince & Williams (2002) linked Globbeae with Zingibereae in subfamily Zingiberoideae based on molecular data. The molecular analysis of Williams et al. (2004) showed that the traditionally recognized genus Mantisia is embedded in Globba, sister to a clade of Indo- Burmese species, a conclusion that is further supported by a number of vegetative and floral characters. Figure 1. Lateral view of mature flower of Globba pendula, an example with two appendages. Abbreviations: a, anther; c, corolla lobe; f, filament; g, ovary; k, calyx lobe; la, labellum; sta, lateral staminode; sti, stigma. Scale bar = 1 mm. The most important character shared by all Globbeae is a unilocular ovary with parietal placentation, as opposed to a trilocular ovary with axile placentation in most of the family (Schumann, 1904; Larsen et al., 1998). However, this distinction is not absolute as some genera of Zingiberaceae have an intermediate placentation type (e.g. Scaphochlamys Baker: Kirchoff 1998). Zingiberaceae are among the most diverse families of monocots in terms of floral morphology and pollination systems (Endress, 1994; Specht et al., 2012). Flowers of all Zingiberaceae are characterized by a single prominent fertile stamen with a broad flattened anther that encloses the style. Globba and Gagnepainia are distinctive in Zingiberaceae in having a large arching filament linked to a small labellum (Fig. 1; Williams et al., 2004; Leong- Škorničková & Newman, 2015). In Globba the filament is greatly elongated and is as long as, or longer than the floral tube and staminodes. The anthers of most Globbaspp. are unique in that they bear two to four, well-developed appendages along the sides of the anthers (Williams et al., 2004). The reported number of anther appendages (zero, two or four) is highly diagnostic and has been used extensively in the infrageneric classification of Globba (Horaninow, 1862; Schumann, 1904; Larsen, 1972; Williams et al., 2004). Table 1. Source of investigated species with reference to the figures RBGE Living collection Plant origin and collection Number of Subgenus Section* Species Figures accession voucher** appendages numbers Mantisia Haplanthera Globba racemose Sm. 19920039 Nepal 0 Figs 2A, 3A–H Mantisia Haplanthera G. racemosaSm. 19991625 Thailand 0 Figs 2B, 3I–L Mantisia Haplanthera G. sessilifloraSims ----- Williams et al. (2004) 0 their Fig. 3 Mantisia Mantisia G. spathulate Roxb. ----- Box & Rudall (2006) 2 their Fig. 6E G. pendula Roxb. Thailand, Nakhon Si (synonym G. Thammarat - Newman & Ceratanthera Ceratanthera fasciata Ridl.) 19860743 Škorničková 1992 2 Fig. 2E, 4G–L Vietnam, Dong Nai Prov. Ceratanthera Ceratanthera G. pendula Roxb. 20081102 - Newman & Poulsen 2435 2 Fig. 1, 2F Ceratanthera Ceratanthera G. geoffrayi Gagnep. 20100754 Cambodia 4 Fig. 2H, 5A–F Indonesia, Aceh - Newman & Globba Nudae G. paniculate Valeton 20010323 Škorničková 2047 2 Fig. 2C, 4A–F G. campsophylla Philippines, Luzon - Newman Globba Nudae K.Schum. 19972553 & Škorničková 2006 2 Fig. 2D G. albiflora Ridl. Laos, Bolikhamxai Globba Nudae var. aurea Holttum 20060805 - Newman et al. LAO 1529 2 Fig. 2G G. flagellaris K. Thailand - Newman & Globba Nudae Larsen 19991624 Škorničková 2048 4 Fig. 2K, L Globba Globba G. candidaGagnep. 20091380 Laos 4 Fig. 2M, N G. siamensis (Hemsl.) Laos, Bolikhamxai Globba Globba Hemsl. 20060813 - Newman et al. LAO1533 6 Fig. 2Q, 6A–H Malaysia, Selangor/ Pahang border - Newman & Globba Sempervirens G. patens Miq. 19680620 Škorničková 2017 4 Fig. 2P Globba Sempervirens G. propinqua Ridl. 19841210 Malaysia, Sabah. 6 Fig. 2R, S Malaysia, Sarawak - Sinclair G. atrosanguinea & Newman & Škorničková Globba Sempervirens Teijsm. & Binn. 19820784 2019 6 Fig. 2T, 5I–P Globba Sempervirens G. cernua Baker ----- Box & Rudall (2006) 4 their Figs 1, 2I Hemiorchis burmanica Chittagong, Bangladesh ----- ------ Kurz 19991652 - Newman 1001 0 Fig. 7E, F Gagnepainia godefroyi Thailand - Tan 5, 15, 16, ----- ----- (Baill.) K.Schum. 20010444 Newman 0 Fig. 7C, D Gagnepainia ----- ----- harmandii K.Schum. 19991163 Thailand, Newman 931 0 Fig. 7A, B Cautleya spicata (Sm.) ----- ----- Baker ----- Ngamriabsakul (2005) 0 his Figs 3, 7, 9, 11 *According to Williams et al. (2004); **refers to herbarium voucher at RBGE (E) when available Floral developmental investigations are known to provide useful characters for phylogenetic studies (e.g. Kirchoff, 1988, 1998; Ronse De Craene & Smets, 1999; Ronse De Craene et al., 1998; Bachelier, Endress & Ronse De Craene, 2011; Prenner & Cardoso, 2017). The primary goal of this study is to document the structure and development of the appendages of Globba, and explore their origins and evolution. The findings of this investigation, combined with results of previous phylogenetic studies, will provide insight into the evolution of Globbeae and shed light on the evolution of developmental patterns. This study sheds particular light on (1) the homology of the appendages in two- and four-appendaged species and (2) the evolution of the various numbers of appendages in the genus. We also provide the first report of six- appendaged Globba spp. MATERIAL AND METHODS Fresh flowers of 15 Globba spp., two Gagnepainia spp. [G. godefroyi (Baill.) K.Schum. and G. harmandii K.Schum.] and Hemiorchis burmanica Kurz were collected by LRDC from the living collections in the Royal Botanic Garden Edinburgh, fixed and stored in 70% ethanol (Table 1). For examination of anther appendages using light microscopy (LM), we dissected mature flower buds in 70% ethanol under a Zeiss Stemi SV6 dissecting microscope. At least five flowers were investigated per taxon. Photomicrographs were taken using a Zeiss Axiophot photomicroscope fitted with a Zeiss Axio-Cam digital camera. For examination of the development of anther appendages using scanning electron microscopy (SEM), we dissected floral buds in 70% ethanol under a Zeiss Stemi SV6 dissecting microscope. After being washed repeatedly in 70% ethanol, floral buds of different